CN111233873B - 甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用 - Google Patents
甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用 Download PDFInfo
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Abstract
本发明公开了甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用，不仅能诱导CHRNA7高表达的AML细胞系KG‑1和HL‑60凋亡，而且能诱导其分化，可以作为治疗初诊以及复发难治性AML的有效药物。
Description
技术领域
本发明涉及一种白血病治疗药物，具体地说是甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用，属于医学技术领域。
背景技术
急性髓系白血病（acute myeloid leukemia,AML）是一种骨髓造血干细胞恶性增殖形成的恶性肿瘤，其发病率和死亡率分别约占总白血病的35%和49%。2015年的中国肿瘤调查数据显示，在我国白血病的死亡率在所有肿瘤中排第9位，而其中主要是除M3以外的其他类型的AML患者（郑荣寿，孙可欣，张思维，曾红梅，邹小农，陈茹，顾秀瑛，魏文强，赫捷.2015年中国恶性肿瘤流行情况分析[J].中华肿瘤杂志,2019,41(1):19-28）。AML可发生于任何年龄，起病急骤，进展迅速，自然存活期在6个月-1年以内。当前，联合化疗仍是AML治疗最常用的手段，但AML患者化疗耐药及在停药后容易复发是影响AML治疗效果的关键因素之一。另有研究发现，白血病干细胞是AML患者化疗耐药及复发的根源（Zeijlemaker W, GrobT, Meijer R, Hanekamp D, Kelder A, Carbaat-Ham JC, Oussoren-Brockhoff YJM,Snel AN. CD34+CD38− leukemic stem cell frequency to predict outcome in acutemyeloid leukemia. Leukemia, 2019, 33(5):1102-1112. ）。因此，启动白血病干细胞凋亡或/与分化通路将更可能治愈AML。寻找一种既能抑制白血病细胞增殖又能抑制白血病干细胞增殖的药物已成为科学家共同追求的目标。
发明内容
为了解决上述问题，本发明提供了甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用，本发明中涉及的甲基牛扁亭不仅可以用于初诊AML，而且可以用于治疗复发难治性AML，并有望治愈AML。
本发明的技术方案为：
甲基牛扁亭在制备抑制白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用。
甲基牛扁亭（Methyllycaconitine，MLA）是来源于中药翠雀种子的一种提取物，其化学式为：
为CHRNA7（α7 nicotinic acetylcholine receptor，尼古丁乙酰胆碱受体α7）的特异性抑制剂，其在CHRNA7中的结合位点与α-金球蛇毒素相似，低剂量时可提高动物的认知功能，并能抑制小胶质细胞释放TNF-α。甲基牛扁亭不仅诱导了CHRNA7高表达的AML细胞系KG-1和HL-60凋亡，而且诱导其分化。同时，浓度高达50μM的甲基牛扁亭对正常人PBMCs存活率没有影响。
应用CCK-8试验计算得到的KG-1和HL-60这2个细胞系经MLA处理96h时的IC50（半抑制浓度）值分别为22.24μM和13.72μM。以IC50为参照，进行后续的细胞凋亡试验和细胞分化试验，MLA处理细胞的时间均采用96h。因此，能够作为治疗初诊以及复发难治性AML的有效药物。
根据动物实验结果推知，MLA应用于人的剂量约为1.1mg/kg，采用静脉途径给药。在无菌条件下抽取所需剂量的浓度为110mg/ml MLA，置于无菌无致热源的含0.9%生理盐水的输液袋中，稀释后的MLA浓度为1.1mg/kg,轻轻颠倒注射袋使溶液混合。静脉注射前观察注射液有无微粒或变色。
本发明的有益效果为：能够作为治疗初诊以及复发难治性AML的有效药物。
下面结合附图和实施例对本发明作进一步说明。
附图说明
图1为本发明实施例CHRNA7在AML细胞系KG-1、HL-60和K562中的表达图；
图2为本发明实施例MLA诱导CHRNA7高表达的AML细胞系KG-1和HL-60细胞凋亡的流式结果图；
图3为本发明实施例MLA诱导KG-1和HL-60细胞凋亡统计结果图
图4为本发明实施例MLA诱导KG-1和HL-60细胞分化瑞氏染色结果图；
图5为本发明实施例MLA诱导KG-1和HL-60细胞分化流式检测结果图；
图6为本发明实施例CHRNA7在AML患者骨髓来源的白血病干细胞中的表达图；
图7为本发明实施例MLA诱导AML白血病干细胞凋亡的流式结果图；
图8为本发明实施例MLA诱导AML白血病干细胞凋亡统计结果图；
图9为本发明实施例MLA诱导AML白血病干细胞分化瑞氏染色结果图；
图10为本发明实施例MLA诱导AML白血病干细胞分化流式检测结果图；
图11为本发明实施例小鼠Kaplan‐Meier生存曲线。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例1
一种用于抑制白血病细胞及白血病干细胞增殖并诱导其分化的小分子，所述小分子为甲基牛扁亭。
甲基牛扁亭（Methyllycaconitine，MLA）是来源于中药翠雀种子的一种提取物，其化学式为：
甲基牛扁亭为CHRNA7（α7 nicotinic acetylcholine receptor，尼古丁乙酰胆碱受体α7）的特异性抑制剂，其在CHRNA7中的结合位点与α-金球蛇毒素相似，低剂量时可提高动物的认知功能，并能抑制小胶质细胞释放TNF-α。实验证实，甲基牛扁亭不仅诱导了CHRNA7高表达的AML细胞系KG-1和HL-60凋亡，而且诱导其分化。同时，证实，浓度高达50μM的甲基牛扁亭对正常人PBMCs存活率没有影响。
实验例一
MLA对白血病细胞及白血病干细胞的杀伤效果实验
（1）白血病细胞系的培养
用含10%胎牛血清的RPMI 1640培养基培养细胞，待其进入对数生长期时收集细胞并计数后，再进行后续实验，包括细胞增殖试验、细胞凋亡试验、细胞分化试验。
（2）急性髓系白血病干细胞的分离与培养
收集AML患者骨髓标本，用PBS等体积稀释，混匀后加入到适量的Ficoll淋巴细胞分离液上方，2000rpm离心25min,吸出单个核细胞，用PBS洗涤2次，即得骨髓单个核细胞；在该细胞标本中，加入鼠抗人CD34-PE和CD38-FITC抗体，用流式细胞分选仪从中分选出CD34+CD38-的细胞，即为白血病干细胞；该细胞可用于MLA抗白血病效果的评价。
（3）CCK8法检测细胞增殖
实验分为2大组：DMSO组和MLA组。每大组设6个不同的剂量，每个剂量做3个复孔。接种细胞至96孔板，5000个细胞/孔，终体积为100μL/孔，于37℃、5% CO2中分别培养24h、48h、72h，于培养结束前4h内向每孔内加入10μL CCK-8，将培养板置于培养箱内孵育2-4小时，用酶标仪测定OD450nm，计算IC50，选择IC50最小值所对应的处理时间作为后续实验的处理时间。
（4）流式细胞术检测细胞凋亡
将处于对数生长期的KG-1、HL-60、K562细胞或从AML患者骨髓中分离得到的白血病干细胞（CD34+CD38-）接种到6孔板中，加入MLA，同时设立空白对照及复孔，于37℃、5%CO2中培养72h后，收集细胞，用结合缓冲液洗涤，分别加入5μL FITC-Annexin V和PI，避光孵育15min, 上流式细胞仪检测，从而确定细胞凋亡情况。
（5）对分化的影响
①瑞氏-吉姆萨染色法：将处于对数生长期的KG-1、HL-60、K562细胞或从AML患者骨髓中分离得到的白血病干细胞（CD34+CD38-）接种到6孔板中，加入MLA，同时设立空白对照及复孔，于37℃、5% CO2中培养96h后，收集细胞，用瑞氏-吉姆萨染液染色1min后，再加入等量的瑞氏-吉姆萨缓冲液，混匀后染色10-15min，光学显微镜下观察细胞分化情况；
②FCM：按照①中的方法处理并收集细胞，每个样品中加入5μL 人FITC-CD14、FITC-CD15抗体，冰上孵育30min，用PBS洗涤1次后上流式细胞仪检测，检测细胞分化情况。
通过上述五个步骤，观察MLA处理前后白血病细胞及白血病干细胞的凋亡与分化情况，确定MLA对白血病细胞及白血病干细胞的杀伤效果。
实验例二
CHRNA7在AML细胞系KG-1、HL-60和K562中的表达
具体方法如下：
CHRNA7在AML细胞系KG-1、HL-60和K562中的表达图（流式细胞术法），如图1所示。
材料与试剂：兔抗人CHRNA7抗体（购自Proteintech公司）、胎牛血清、含3%胎牛血清的PBS，Alexa Fluor647标记的山羊抗兔IgG二抗
操作步骤：用含10%%胎牛血清的RPMI 1640培养基培养KG-1、HL-60、K562细胞，待其进入对数生长期时，收集细胞，于1000rpm离心5min，弃上清，在细胞沉淀中加入1ml 含3%胎牛血清的PBS，重悬细胞，于1000rpm离心5min，弃上清，细胞计数，从中去除1×106个细胞，加入10μL 兔抗人CHRNA7抗体（Proteintech，1:2000稀释），冰上孵育1h后，用含3%胎牛血清的PBS 洗涤2次，弃上清，加入Alexa Fluor647标记的山羊抗兔IgG二抗，冰上孵育30min，加入1ml含3%胎牛血清的PBS重悬细胞，1000rpm离心5min，弃上清，以仅加AlexaFluor647标记的山羊抗兔IgG二抗组（isotype组）以及不加任何抗体组（ctrl组）作为对照，上流式细胞仪进行检测。
实验结果：从图1中可以看出，实验组中携带荧光的KG-1细胞所占的百分率为94%，而ctrl组和isotype组中携带荧光的KG-1细胞所占的百分率仅为0.01%和7.71%，所以实验组中携带荧光的KG-1细胞百分率明显高于对照组，故KG-1细胞表面CHRNA7蛋白高表达。同样的，实验组中携带荧光的HL-60细胞百分率（98.82%）明显高于对照组（0.67%和4.16%），故HL-60细胞表面CHRNA7蛋白也高表达；而实验组中携带荧光的K562细胞百分率（2.16%）明显高于对照组（0.09%和3.91%），故K562细胞表面CHRNA7蛋白不表达。
实验例三
MLA诱导CHRNA7高表达的AML细胞系KG-1和HL-60凋亡及分化情况，如图2、图3、图4和图5所示。
材料与试剂：MLA溶液（用DMSO溶解成终浓度为10mM，购自MCE）,RPMI 1640培养基、胎牛血清、Annexin-V-FITC/PI细胞凋亡试剂盒（购自BD Bioscience）、FITC标记的鼠抗人CD14抗体和CD15抗体（购自BD Bioscience）、15ml无菌离心管、1.5ml离心管、10μL、200μL和1mL无菌吸头、6孔细胞培养板。
操作步骤：用含10%%胎牛血清的RPMI 1640培养基培养KG-1和HL-60细胞，待其进入对数生长期时收集细胞并计数，接种于6孔板中，50×106/孔，加入MLA，使其终浓度为5μM和10μM，以加DMSO组为对照，于37℃、5%CO2的细胞培养箱中培养96h，然后收集细胞，将其分为3份，其中第一份细胞于1000rpm离心5min,弃上清，在细胞沉淀中加入1ml 1×BindingBuffer, 重悬细胞沉淀，分别加入5μL Annexin-V-FITC和PI，于冰上避光染色15min,用流式细胞仪进行检测；第二份细胞于1000rpm离心5min,弃上清，涂片，晾干后用瑞氏-吉姆萨染液染色1min后，再加入等量的瑞氏-吉姆萨缓冲液，混匀后染色10-15min，光学显微镜下观察细胞分化情况；第三份细胞于1000rpm离心5min,弃上清，加入1ml含3%胎牛血清的PBS，平均分成2管，1000rpm离心5min,弃上清，分别加入5μL 鼠抗人CD14-FITC和CD15-FITC抗体，冰上孵育30min，加入1ml含3%胎牛血清的PBS重悬细胞，1000rpm离心5min，弃上清。
实验结果：从图2和图3的结果中可以看出，当MLA浓度为5μM和10μM时，KG-1细胞凋亡率分别为27.5%±1.37%和42.39%±0.99%，明显高于不加药组（12.68%±1.36%）；HL-60细胞凋亡率分别为25.72%±1.42%和35.16%±1.22%，明显高于不加药组（11.64%±1.52%，P>0.05）。从图4结果可以看出，与未加药组相比，加MLA组中部分KG-1细胞核分叶增多，部分细胞核质比下降；而在HL-60细胞中，核分叶增多的细胞也明显增多。图5的流式检测结果表明，与未加药组相比，在加入FITC标记的鼠抗人CD14抗体后，加MLA组的KG-1细胞荧光强度明显增强，说明MLA能诱导KG-1细胞表面的成熟单核细胞标志CD14的表达；同时，与未加药组相比，在加入FITC标记的鼠抗人CD15抗体后，加MLA组的HL-60细胞荧光强度明显增强，说明MLA能诱导HL-60细胞表面的成熟髓系细胞标志CD15的表达。
通过以上数据可以看出，MLA不仅诱导了CHRNA7高表达的AML细胞系KG-1和HL-60凋亡（图2、图3），而且诱导其分化（图4、图5）。
实验例四
CHRNA7在白血病干细胞中的表达情况，如图6所示。
材料与试剂：Ficoll淋巴细胞分离液、PBS、鼠抗人CD34和CD38抗体、FITC标记的鼠抗人CD38抗体和PE标记的鼠抗人CD34抗体（购自BD Bioscience）、兔抗人CHRNA7抗体、Alexa Fluor647标记的山羊抗兔IgG二抗、15ml无菌离心管、1.5ml离心管、10μL、200μL和1mL无菌吸头。
操作步骤：
1、骨髓单个核细胞的分离：收集急性髓系白血病患者骨髓,用等量PBS稀释，加入到Ficoll淋巴细胞分离液上层，2000rpm离心20min，吸出单个核细胞层加入到一新的离心管中，加入适量的含3%胎牛血清的PBS，1500rpm离心5min后，弃上清，用含3%胎牛血清的PBS重悬细胞并计数。
2、CD34+CD38-细胞的分选：向1中分离得到的人骨髓单个核细胞中加入鼠抗人CD34抗体，每106细胞中加入5μL，充分混匀细胞，冰上孵育30min,用细胞分选仪（BD FACSARIAII）进行分选，获得CD34+CD38-细胞，即为白血病干细胞（LSCs）。
3、CHRNA7在白血病干细胞表面的表达情况：从2中分离得到的细胞中取出100万个细胞平均分成3份：在第一份细胞中加入兔抗人CHRNA7抗体，冰上孵育2h,用含3%胎牛血清的PBS洗涤2次后，加入2μL Alexa Fluor647标记的山羊抗兔IgG二抗、FITC标记的鼠抗人CD38抗体和PE标记的鼠抗人CD34抗体，冰上孵育30min，加入1ml 含3%胎牛血清的PBS，于1000rpm离心5min后，弃上清，上流式细胞仪进行检测。在第二份细胞中加入FITC标记的鼠抗人CD38抗体，冰上孵育30min，加入1ml 含3%胎牛血清的PBS，于1000rpm离心5min后，弃上清，上流式细胞仪进行检测。在第三份细胞中PE标记的鼠抗人CD34抗体，冰上孵育30min，加入1ml 含3%胎牛血清的PBS，于1000rpm离心5min后，弃上清，上流式细胞仪进行检测。
实验结果：从图6结果可以看出，白血病干细胞即CD34+CD38-细胞中CHRNA7为阳性的细胞约占68.62%，说明CHRNA7在白血病干细胞中高表达。
实验例五
MLA对白血病干细胞凋亡的影响，如图7、图8、图9和图10所示。
材料与试剂：MLA溶液（用DMSO溶解成终浓度为10mM，购自MCE）、RPMI 1640培养基、胎牛血清、Annexin-V-FITC/PI细胞凋亡试剂盒（购自BD Bioscience）、FITC标记的鼠抗人CD14抗体和CD15抗体（购自BD Bioscience）、15ml无菌离心管、1.5ml离心管、10μL、200μL和1mL无菌吸头、12孔细胞培养板。
操作步骤：将分选得到的白血病干细胞接种到12孔板中， 50×106/孔，加入MLA，使其终浓度分别为5μM和10μM，以加DMSO组为对照，于37℃、5%CO2的细胞培养箱中培养96h，然后收集细胞，将其分为3份，其中第一份细胞于1000rpm离心5min,弃上清，在细胞沉淀中加入1ml 1×Binding Buffer, 重悬细胞沉淀，分别加入5μL Annexin-V-FITC和PI，于冰上避光染色15min,用流式细胞仪进行检测；第二份细胞于1000rpm离心5min,弃上清，涂片，晾干后用瑞氏-吉姆萨染液染色1min后，再加入等量的瑞氏-吉姆萨缓冲液，混匀后染色10~15min，光学显微镜下观察细胞分化情况；第三份细胞于1000rpm离心5min,弃上清，加入1ml含3%胎牛血清的PBS，平均分成2管，1000rpm离心5min,弃上清，分别加入5μL 鼠抗人CD15-FITC抗体，冰上孵育30min，加入1ml含3%胎牛血清的PBS重悬细胞，1000rpm离心5min，弃上清。
实验结果：从图7和图8的结果中可以看出，当MLA浓度为5μM和10μM时，白血病干细胞凋亡率分别为42.22%±4.37%和71.11%±3.99%，明显高于不加药组（13.45%±1.56%）。从图9结果可以看出，与未加药组相比，加MLA组中部分白血病干细胞核分叶增多，部分细胞核质比下降。图10的流式检测结果表明，与未加药组相比，在加入FITC标记的鼠抗人CD15抗体后，加MLA组的白血病干细胞荧光强度明显增强，说明MLA能诱导白血病干细胞表面的成熟髓系细胞标志CD15的表达。
通过以上数据可以看出，MLA不仅诱导了白血病干细胞凋亡（图7、图8），而且诱导其分化（图9、图10）。
实验例五
材料与试剂：MLA溶液（用DMSO溶解成终浓度为50mg，购自MCE），RPMI 1640培养基、胎牛血清、细胞计数板
实验动物：NSG小鼠
操作步骤：用含10%胎牛血清的RPMI 1640培养基培养KG-1细胞，待其进入对数生长期时收集细胞并计数，接种6 ~8周龄NSG小鼠，5×106细胞/只，3天后开始静脉注射给予MLA，10mg/kg，每3天注射1次，每天观察小鼠行为、体重、生存状况等。
实验结果：从图11可以看出，注射MLA组的NSG小鼠平均生存期为63.5天，明显长于空白对照组（平均生存期为34.5天）和溶剂组（平均生存期为34天）（P=0.016）。
Claims (2)
1.甲基牛扁亭在制备抑制急性髓系白血病细胞及白血病干细胞增殖并诱导其分化药物中的应用；
所述甲基牛扁亭的化学式为：
。
2.如权利要求1所述的应用，其特征在于：甲基牛扁亭在制备用以治疗初诊以及复发难治性AML的有效药物中的用途。
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